Bottom Line:
By comparison, a fully-extended conformation was stable.A newly-formed coordination between the α(v) Asp457 and the α-genu metal ion might contribute to the stability of the fully-extended conformation.These results reveal the dynamic processes and pathways of integrin conformational changes with atomic details and provide new insights into the structural mechanisms of integrin activation.

Affiliation: Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, Georgia, United States of America.

ABSTRACTIntegrins may undergo large conformational changes during activation, but the dynamic processes and pathways remain poorly understood. We used molecular dynamics to simulate forced unbending of a complete integrin α(v)β₃ ectodomain in both unliganded and liganded forms. Pulling the head of the integrin readily induced changes in the integrin from a bent to an extended conformation. Pulling at a cyclic RGD ligand bound to the integrin head also extended the integrin, suggesting that force can activate integrins. Interactions at the interfaces between the hybrid and β tail domains and between the hybrid and epidermal growth factor 4 domains formed the major energy barrier along the unbending pathway, which could be overcome spontaneously in ~1 µs to yield a partially-extended conformation that tended to rebend. By comparison, a fully-extended conformation was stable. A newly-formed coordination between the α(v) Asp457 and the α-genu metal ion might contribute to the stability of the fully-extended conformation. These results reveal the dynamic processes and pathways of integrin conformational changes with atomic details and provide new insights into the structural mechanisms of integrin activation.

pcbi-1001086-g002: Forced unbending of unliganded integrin αVβ3.A. U1 in the enlarged water box for unbending simulations. B. Illustration of force application on the head and constraint on the βTD in the SMD simulations of U1 and U2. C. Snapshots of a representative unbending process (U1 SMD 1) taken at indicated times and extensions. D. Force-extension curves in the constant-velocity SMD simulations of U1 by pulling the βA and/or β-propeller domains with a 2 nm ns−1 pulling speed and a 0.5 kcal mol−1 Å−2 spring constant. E. Force-extension curves in the constant-velocity SMD simulations of U1 by pulling the βA domain with indicated pulling speeds and spring constants. F. Force-extension curves for three constant-velocity SMD simulations of U1 and one constant-velocity SMD simulation of U2 with a 2 nm ns−1 pulling speed and a 0.5 kcal mol−1 Å−2 spring constant. Red and blue circles indicate respective structures along the unbending pathways from the trajectories of the U1 SMD 1 & 2 that were selected as starting structures for free MD simulations. The left two represent partially-extended structures and the right two represent fully-extended structures. The red curves in panels D–F are all for the U1 SMD 1.

Mentions:
We used constant-velocity SMD simulations [25] to accelerate integrin unbending. The enlarged systems with bigger water boxes to accommodate unbending (Fig. 2A) were first equilibrated using 4–8 ns free dynamics. A force was then applied to the head of the unliganded integrin αVβ3 (U1 or U2), where ligand-binding sites are, by a spring moving at a constant velocity while the βTD was constrained to mimic the membrane anchorage (Fig. 2B). The α-leg was not constrained to allow separation of two legs, should it occur. Regardless of the initial pulling directions, force became aligned along the direction from the constraint location to the pulling location after the integrin underwent a rigid-body rotation. Remarkably, pulling readily caused gradual unbending of integrin αVβ3 without major distortions of its individual domains, resulting in a fully-extended conformation with a closed headpiece and closed legs (Fig. 2C and Videos S1 & S2).

pcbi-1001086-g002: Forced unbending of unliganded integrin αVβ3.A. U1 in the enlarged water box for unbending simulations. B. Illustration of force application on the head and constraint on the βTD in the SMD simulations of U1 and U2. C. Snapshots of a representative unbending process (U1 SMD 1) taken at indicated times and extensions. D. Force-extension curves in the constant-velocity SMD simulations of U1 by pulling the βA and/or β-propeller domains with a 2 nm ns−1 pulling speed and a 0.5 kcal mol−1 Å−2 spring constant. E. Force-extension curves in the constant-velocity SMD simulations of U1 by pulling the βA domain with indicated pulling speeds and spring constants. F. Force-extension curves for three constant-velocity SMD simulations of U1 and one constant-velocity SMD simulation of U2 with a 2 nm ns−1 pulling speed and a 0.5 kcal mol−1 Å−2 spring constant. Red and blue circles indicate respective structures along the unbending pathways from the trajectories of the U1 SMD 1 & 2 that were selected as starting structures for free MD simulations. The left two represent partially-extended structures and the right two represent fully-extended structures. The red curves in panels D–F are all for the U1 SMD 1.

Mentions:
We used constant-velocity SMD simulations [25] to accelerate integrin unbending. The enlarged systems with bigger water boxes to accommodate unbending (Fig. 2A) were first equilibrated using 4–8 ns free dynamics. A force was then applied to the head of the unliganded integrin αVβ3 (U1 or U2), where ligand-binding sites are, by a spring moving at a constant velocity while the βTD was constrained to mimic the membrane anchorage (Fig. 2B). The α-leg was not constrained to allow separation of two legs, should it occur. Regardless of the initial pulling directions, force became aligned along the direction from the constraint location to the pulling location after the integrin underwent a rigid-body rotation. Remarkably, pulling readily caused gradual unbending of integrin αVβ3 without major distortions of its individual domains, resulting in a fully-extended conformation with a closed headpiece and closed legs (Fig. 2C and Videos S1 & S2).

Bottom Line:
By comparison, a fully-extended conformation was stable.A newly-formed coordination between the α(v) Asp457 and the α-genu metal ion might contribute to the stability of the fully-extended conformation.These results reveal the dynamic processes and pathways of integrin conformational changes with atomic details and provide new insights into the structural mechanisms of integrin activation.

Affiliation:
Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, Georgia, United States of America.

ABSTRACTIntegrins may undergo large conformational changes during activation, but the dynamic processes and pathways remain poorly understood. We used molecular dynamics to simulate forced unbending of a complete integrin α(v)β₃ ectodomain in both unliganded and liganded forms. Pulling the head of the integrin readily induced changes in the integrin from a bent to an extended conformation. Pulling at a cyclic RGD ligand bound to the integrin head also extended the integrin, suggesting that force can activate integrins. Interactions at the interfaces between the hybrid and β tail domains and between the hybrid and epidermal growth factor 4 domains formed the major energy barrier along the unbending pathway, which could be overcome spontaneously in ~1 µs to yield a partially-extended conformation that tended to rebend. By comparison, a fully-extended conformation was stable. A newly-formed coordination between the α(v) Asp457 and the α-genu metal ion might contribute to the stability of the fully-extended conformation. These results reveal the dynamic processes and pathways of integrin conformational changes with atomic details and provide new insights into the structural mechanisms of integrin activation.